Compositions and methods for treating viral infections in shrimps

Abstract

Nanoparticles comprising partially deacetylated chitosan and ss or dsRNA partially complementary to, binding to or at least 90% identical to mRNA targets of viruses pathogenic in farmed crustaceans, compositions and farmed aquatic crustaceans comprising the same, and methods for their use in treating or preventing viral infection in aquaculture are provided.

What is claimed is: 
     
       1. A chitosan-RNA nanoparticle comprising partially deacetylated chitosan and at least one RNA sequence at least 50 bases in length, wherein the degree of deacetylation of said chitosan is in the range of 42% to 35%, and wherein said RNA sequence is capable of silencing expression of a gene when administered to an organism expressing said gene. 
     
     
       2. The chitosan-RNA nanoparticle of  claim 1 , wherein said degree of deacetylation of said chitosan is 35%. 
     
     
       3. The chitosan-RNA nanoparticle of  claim 1 , wherein said at least one RNA oligonucleotide is a single stranded RNA (ssRNA). 
     
     
       4. The chitosan-RNA nanoparticle of  claim 1 , wherein said at least one RNA oligonucleotide is a double-stranded RNA (dsRNA). 
     
     
       5. The chitosan-RNA nanoparticle of  claim 2 , wherein said at least one RNA oligonucleotide is a double-stranded RNA (dsRNA). 
     
     
       6. A nutraceutical composition comprising farmed crustacean food and the chitosan-RNA nanoparticle of  claim 1 . 
     
     
       7. A farmed crustacean comprising the nanoparticle of  claim 1 . 
     
     
       8. A method of treatment or prevention of a disease or condition in a farmed crustacean associated with a pathogenic virus, the method comprising feeding the farmed crustacean the nanoparticle of  claim 1 , wherein said at least one RNA sequence is targeted to a gene product of said pathological virus, and wherein ingestion of said nanoparticle by said farmed crustacean results in reduction in the level of a pathological virus in said farmed crustacean, compared to the same farmed crustacean ingesting feed devoid of RNA targeted to a gene product of said pathological virus. 
     
     
       9. The chitosan-RNA nanoparticle of  claim 1 , wherein said at least one RNA sequence comprises one or more of the following:
 (a) at least one sequence capable of binding through complementary base pairing to a target mRNA molecule of a virus pathogenic in farmed crustaceans; 
 (b) at least one sequence having at least 90% sequence identity to a target mRNA molecule of a virus pathogenic in farmed crustaceans; 
 (c) at least one sequence at least partially complementary to a target mRNA molecule of a virus pathogenic in farmed crustaceans; 
 (d) at least one sequence identical to at least 20 contiguous bases of a target mRNA molecule of a virus pathogenic in farmed crustaceans; 
 (e) at least one sequence capable of binding through complementary base pairing to a target mRNA molecule of a farmed crustacean; 
 (f) at least one sequence identical to at least 20 contiguous bases of a target mRNA molecule of a farmed crustacean, and 
 (g) at least one sequence at least partially complementary to a target mRNA molecule of a farmed crustacean. 
 
     
     
       10. The method of  claim 8 , wherein the degree of deacetylation of said chitosan is 35%. 
     
     
       11. The method of  claim 8 , wherein said farmed crustaceans are selected from the group consisting of Shrimp, Prawns, Crabs, Lobsters and Crayfishes. 
     
     
       12. The method of  claim 8 , wherein said farmed crustaceans are shrimp or prawns. 
     
     
       13. The method of  claim 8 , wherein said virus is selected from the group consisting of White Spot Syndrome Virus (WSSV), Taura syndrome virus (TSV), Yellow head virus (YHV), Gill-associated virus (GAV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Infectious myonecrosis virus (IMNV), White Tail Disease ( Macrobracium rosenbergii  nodavirus, MrNV) and Infectious pancreatic necrosis virus (IPNV). 
     
     
       14. The chitosan-RNA nanoparticle of  claim 9 , wherein said partially complementary sequence or sequence capable of binding through complementary base pairing to said target nucleic acid molecule of said at least one RNA sequence is complementary to a sequence of said target nucleic acid molecule at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 400, at least 500, at least 600 and at least 750 bases in length. 
     
     
       15. The chitosan-RNA nanoparticle of  claim 9 , wherein said at least one RNA sequence comprising at least one sequence identical to at least 21 contiguous bases of a target mRNA molecule of said pathogenic virus in farmed crustaceans is identical to at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 400, at least 500, at least 600 and at least 750 bases of said target mRNA molecule. 
     
     
       16. The chitosan-RNA nanoparticle of  claim 9 , wherein said RNA comprising at least one sequence capable of binding through complementary base pairing to a target mRNA molecule of a virus pathogenic in farmed crustaceans or comprising at least one sequence at least partially complementary to a target mRNA molecule of a virus pathogenic in farmed crustaceans comprises:
 (a) a nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 and 5, or 
 (b) a nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59, 64, 77, 78 and 79. 
 
     
     
       17. The chitosan-RNA nanoparticle of  claim 9 , wherein said RNA comprising at least one sequence having at least 90% sequence identity to a target mRNA molecule of a virus pathogenic in farmed crustaceans or
 at least one sequence identical to at least 20 contiguous bases of a target mRNA molecule of a virus pathogenic in farmed crustaceans comprises: 
 (a) a nucleic acid sequence identical to at least 21 contiguous bases of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 and 5, or 
 (b) a nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59, 64, 77, 78 and 79. 
 
     
     
       18. The chitosan-RNA nanoparticle of  claim 9 , wherein said target mRNA molecule of said farmed crustacean comprises a Rab7 mRNA sequence or fragments thereof. 
     
     
       19. The chitosan-RNA nanoparticle of  claim 18 , wherein said chitosan-RNA nanoparticle comprises an RNA sequence selected from the group consisting of SEQ ID NOs: 47, 53, 54. 
     
     
       20. The chitosan-RNA nanoparticle of  claim 9 , comprising at least one additional RNA sequence capable of directing cleavage of a target mRNA molecule of a virus pathogenic in farmed crustaceans. 
     
     
       21. The chitosan-RNA nanoparticle of  claim 20 , wherein said at least one RNA sequence and said at least one additional RNA sequence are directed to a target mRNA of the same virus. 
     
     
       22. The chitosan-RNA nanoparticle of  claim 20 , wherein said at least one RNA sequence and said at least one additional RNA sequence are directed to a target mRNA of different viruses. 
     
     
       23. The chitosan-RNA nanoparticle of  claim 1 , wherein the particle size of said nanoparticle is in the range of 50-500 nm, as measured by effective z-average diameter. 
     
     
       24. The chitosan-RNA nanoparticle of  claim 1 , wherein said chitosan is conjugated to a molecule selected from the group consisting of biotin, glucuronic acid and a polymer. 
     
     
       25. The chitosan-RNA nanoparticle of  claim 1 , wherein the particle size of said nanoparticle is in the range of 100-200 nm, as measured by effective z-average diameter. 
     
     
       26. The method of  claim 10 , wherein said virus is selected from the group consisting of White Spot Syndrome Virus (WSSV), Taura syndrome virus (TSV), Yellow head virus (YHV), Gill-associated virus (GAV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Infectious myonecrosis virus (IMNV), White Tail Disease ( Macrobracium rosenbergii  nodavirus, MrNV) and Infectious pancreatic necrosis virus (IPNV). 
     
     
       27. The chitosan-RNA nanoparticle of  claim 9 , wherein said RNA comprising at least one sequence capable of binding through complementary base pairing to a target mRNA molecule of a virus pathogenic in farmed crustaceans or comprising at least one sequence at least partially complementary to a target mRNA molecule of a virus pathogenic in farmed crustaceans comprises:
 (a) a nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 and 5, or 
 (b) a nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59, 64, 77, 78 and 79, 
 and wherein the degree of deacetylation of said chitosan is 35%. 
 
     
     
       28. The chitosan-RNA nanoparticle of  claim 9 , wherein said RNA comprising at least one sequence having at least 90% sequence identity to a target mRNA molecule of a virus pathogenic in farmed crustaceans or
 at least one sequence identical to at least 20 contiguous bases of a target mRNA molecule of a virus pathogenic in farmed crustaceans comprises: 
 (a) a nucleic acid sequence identical to at least 21 contiguous bases of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 and 5, or 
 (b) a nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 59, 64, 77, 78 and 79, 
 and wherein the degree of deacetylation of said chitosan is 35%.